Immature erythrocytes in the blood are called reticulocytes, and normally account for 0.7-2.2% of the total count of erythrocytes. Determination of reticulocyte count helps confirmation of the diagnosis of such diseases as acute internal hemorrhage, hemolytic anemia, aplastic anemia, etc., and also helps in monitoring the progress of a patient's conditions after drug administration; thus it is regarded as being very important in the field of clinical laboratory tests.
A method which has been employed for the counting of said reticulocytes is conducted as follows: a smear of blood sample stained with a basic dye such as new methylene blue, brilliant cresyl blue, etc. is observed and the percentage of the count of stained reticulocytes is determined with respect to the total erythrocyte count.
This method requires a lot of time and involves a considerable workload for pre-treatment of blood samples, e.g. staining, etc., and as well as for visual counting after staining, and is inappropriate when the number of samples is large.
Therefore, many methods have been proposed in which reticulocyte counting is automated by the application of flow cytometry. For example, methods in which a fluorochrome reagent containing Auramine O is used for counting reticulocytes by flow cytometry are disclosed in Japanese Patent Public Disclosure No. 280565/1986 and Japanese Patent Public Disclosure No. 34058/1987.
The present inventors conducted reticulocyte counting by flow cytometry in the following way using a reagent of the composition shown below as an example of a staining solution, in order to examine the effects of Auramine O described in the aforementioned prior literature, Japanese Patent Public Disclosure No. 280565/1986 and Japanese Patent Public Disclosure No. 34058/1987:
______________________________________ Auramino O 500 ppm ethylene glycol 2.5 v/v % HEPES .multidot. sodium hydroxide 20 mM N-2-hydroxyethylpiperazine-N'-2 ethane sulfonic acid NaCl 120 mM ______________________________________
The measurement was conducted 12 times continuously for each blood sample using a flow cytometry apparatus which was turned to have an appropriate degree of sensitivity. About 20 seconds before each measurement, the blood sample vessel containing the blood sample was repeatedly turned upside down for the purpose of stirring it. The blood sample vessel was opened and 400 .mu.l of the blood sample inside was aspirated immediately before each measurement.
FIG. 1 shows the measurement results for two samples. Both of the samples showed decrease in reticulocyte count (Ret%) as the measurements were repeated. Under conditions like this, accurate reticulocyte counting cannot be expected.
In the above-mentioned measurements, the value of blood required each time was as large as 400 .mu.l because a fully automated flow cytometry apparatus was used. If, however, the blood sample is subjected to flow cytometry after being stained manually, only about 10 .mu.l of blood is necessary. In cases where blood is aspirated by 10 .mu.l a notable change in reticulocyte count (%) such as that observed in FIG. 1 does not occur. Even if non-specific staining occurs and the fluorescence intensity increases as a whole, an almost completely accurate reticulocyte count (%) will be recorded as long as the sensitivity of the apparatus is controlled appropriately for that condition.
In other words, non-specific staining occurs only when a continuous measurement is made with a sample by aspirating a large volume of blood from the same sample each time.